New modes and mechanisms of negative regulation of LIN-12/Notch in C. elegans

 描述(由申请人提供)：本申请的总体目标是阐明Notch信号负调控的不同机制。Noch是动植物发育过程中细胞间相互作用和细胞命运决定的主要信号系统中的同名受体。适当地调节Notch信号--在空间、时间、强度或持续时间--对正常发育至关重要。此外，Notch活性的异常与许多不同的癌症以及发育、免疫和神经疾病有关。因此，这项拟议的工作对人类健康有许多影响。对发育机制的深入理解对于开发人类疾病的诊断和治疗工具具有巨大的潜力，这是美国国立卫生研究院的中心任务。我们过去在线虫LIN-12/Notch信号方面的工作为了解Notch信号的保守作用、机制和调控提供了许多基本的见解。我们希望我们在这个系统中的继续工作将继续揭示普遍相关的机制，并选择实验设计的特定方面来最大化这一前景。本申请的每个目的都探索了在教科书发育范例中调节Lin-12/Notch活性和稳定性的不同方式，在该范例中，六个多潜能前体细胞通过Lin-12/Notch介导的细胞-细胞相互作用采用三种不同的命运之一。该模式是精确和稳健的，反映了对Lin-12/Notch活性的严格的时空控制，以及将Lin-12/Notch活性与其他保守的信号通路整合的多种模式。在目标1中，我们建议表征我们在靶向筛查中发现的新的激酶，以寻找负调控因子和另一个基因，该基因首先通过癌症患者的突变而鉴定，我们显示其在线虫中起负调控作用。为了评估它们在Notch调控中的功能保守性，我们将进行人体细胞测试。在目标2中，我们建议研究LIN-12/Notch内吞转运及其在发育模式中受EGF受体活性的负调控。EGF受体和Notch一样，是一种重要的癌基因，这些通路之间的串扰在癌症和正常发育中都会发生。在目标3中，我们建议阐明EGF受体如何在发育模式中抑制激活的核LIN-12/Notch，以及在安静时如何通过胰岛素/胰岛素样信号来响应不利的环境。静止是对人类健康至关重要的基本属性，允许干细胞在有能力的状态下持续较长时间，以便在细胞因衰老、损伤或疾病而丢失时可用于重新填充组织，对于癌症，静止的干细胞被认为对标准化疗具有抵抗力。总而言之，这些目标将提供许多关于发育和疾病中基本信号通路的负调控的新信息，这与NIH的使命一致。

 DESCRIPTION (provided by applicant): The overall goal of this application is to elucidate different mechanisms for negative regulation of Notch signaling. Notch is the eponymous receptor in a major signaling system for cell-cell interactions and cell fate specification in animl development. Regulating Notch signaling appropriately--in space, time, strength or duration--is critically important for normal development. Furthermore, aberrant Notch activity has been implicated in many different cancers, as well as in developmental, immune, and neurological disorders. Thus, the proposed work has many implications for human health. The deeper understanding of developmental mechanism has great potential for developing diagnostic and therapeutic tools for human disease, a central mission of the NIH. Our past work on LIN-12/Notch signaling in C. elegans has afforded many fundamental insights into conserved roles, mechanisms, and regulation of Notch signaling. We expect that our continued work in this system will continue to reveal mechanisms of general relevance, and particular aspects of experimental design were chosen to maximize this prospect. Each aim of this application explores different ways LIN-12/Notch activity and stability is modulated in a textbook developmental paradigm in which six multipotential precursor cells adopt one of three distinct fates through LIN-12/Notch-mediated cell-cell interactions. The patterning is precise and robust, reflecting tight spatial and temporal control of LIN-12/Notch activity and multiple modes for integrating LIN-12/Notch activity with other conserved signaling pathways. In Aim 1, we propose to characterize new kinases we identified in a targeted screen for negative regulators and another gene, first identified by a mutation in a cancer patient, we showed acts as a negative regulator in C. elegans. To assess their functional conservation in Notch regulation, we will perform a human cell assay. In Aim 2, we propose to investigate LIN-12/Notch endocytic trafficking and its negative regulation by EGF Receptor activity in developmental patterning. EGF Receptor, like Notch, is an important oncogene, and crosstalk between these pathways occurs in cancer as well as in normal development. In Aim 3, we propose to elucidate how activated nuclear LIN-12/Notch is inhibited by EGF receptor during developmental patterning and by Insulin/Insulin-like Signaling during quiescence in response to unfavorable environment. Quiescence is a fundamental property of critical importance for human health, allowing stem cells to persist over prolonged periods in a competent state so as to be available to repopulate tissues when cells are lost to aging, injury or disease, and for cancer, quiescent stem-like cells are believed to be resistant to standard chemotherapy. Together, these aims will provide much new information about negative regulation of a fundamental signaling pathway in development and disease, in accord with the mission of the NIH.